Dogwoods (Cornus spp.) are valuable nursery crops grown as landscape plants throughout much of the world. Although there has been considerable work on breeding and selecting dogwoods, there is little information available on genome sizes (DNA content) and ploidy levels within the genus, particularly for specific clones and cultivars. Our objective was to conduct a survey of relative genome sizes and ploidy levels of dogwood taxa representative of the big-bracted, cornelian cherry, and dwarf dogwood clades. Flow cytometry was used to determine relative genome sizes and ploidy levels of 94 accessions of various species, hybrids, and cultivars. Traditional cytology was performed on root cells of representative taxa to calibrate genome sizes with ploidy level. All dogwood accessions tested were diploid with the exception of C. canadensis that was tetraploid and the hybrid cultivar C. KN30-8 that was triploid. Relative genome sizes varied by clade, subgenus, and species with 1Cx values ranging from 1.07 pg for C. canadensis to 5.08 pg for C. eydeana. Relative genome sizes were also valuable for confirming hybridity of interspecific crosses in cases in which parents varied substantially in relative genome size and hybrids were intermediate. A broad range of interspecific hybrids was documented including C. capitata × florida, C. capitata × kousa, C. elliptica × florida, C. hongkongensis × florida, C. kousa × elliptica, and C. kousa × florida. These results provide further insights into the cytogenetics, reproductive biology, crossability, and relative genome sizes of dogwoods.

Abstract

Dogwoods (Cornus spp.) are valuable nursery crops grown as landscape plants throughout much of the world. Although there has been considerable work on breeding and selecting dogwoods, there is little information available on genome sizes (DNA content) and ploidy levels within the genus, particularly for specific clones and cultivars. Our objective was to conduct a survey of relative genome sizes and ploidy levels of dogwood taxa representative of the big-bracted, cornelian cherry, and dwarf dogwood clades. Flow cytometry was used to determine relative genome sizes and ploidy levels of 94 accessions of various species, hybrids, and cultivars. Traditional cytology was performed on root cells of representative taxa to calibrate genome sizes with ploidy level. All dogwood accessions tested were diploid with the exception of C. canadensis that was tetraploid and the hybrid cultivar C. KN30-8 that was triploid. Relative genome sizes varied by clade, subgenus, and species with 1Cx values ranging from 1.07 pg for C. canadensis to 5.08 pg for C. eydeana. Relative genome sizes were also valuable for confirming hybridity of interspecific crosses in cases in which parents varied substantially in relative genome size and hybrids were intermediate. A broad range of interspecific hybrids was documented including C. capitata × florida, C. capitata × kousa, C. elliptica × florida, C. hongkongensis × florida, C. kousa × elliptica, and C. kousa × florida. These results provide further insights into the cytogenetics, reproductive biology, crossability, and relative genome sizes of dogwoods.

Dogwoods include more than 50 species of shrubs, small trees, and a few herbaceous perennials with distribution that ranges across the northern hemisphere and rarely into the southern hemisphere (Eyde, 1988; Fan and Xiang, 2001; Reed, 2004; Xiang et al., 2006). Many of these species are valuable landscape plants and are frequently cultivated for their four-season attributes including attractive flowers, fruit, foliage, bark, and form (Cappiello and Shadow, 2005). According to the Census of Horticultural Specialties (USDA-NASS, 2010), 2009 sales of dogwoods totaled more than $30.9 million in the United States.

Despite the considerable breeding, selection, and widespread cultivation of dogwoods, sampling for genome size and ploidy level of species, cultivars, and hybrids has been limited and little is known concerning ploidy level and genome size of specific cultivars and hybrids. The objectives of this study were to determine relative genome sizes and ploidy levels of diverse taxa within Cornus, specifically for the BB, CC, and DW clades.

Materials and Methods

Flow cytometry.

Relative 2C genome sizes were determined using flow cytometry (Greilhuber et al., 2007). Plant tissue, including expanding leaf tissue, vegetative buds, and floral buds, was collected from 94 accessions (Table 1) at the Mountain Horticultural Crop Research and Extension Center of North Carolina State University located in Mills River, NC. These included nine species and seven groups of hybrids representing BB, CC, and DW dogwoods that were sampled over time (May 2012 through Mar. 2013) in a completely randomized design. In addition, leaf tissue from the original Cornus ‘KN30-8’ (Venus™) was provided by Dr. Thomas Molnar at Rutgers University. Approximately 1 cm2 or 20 mg of tissue was finely chopped in a polystyrene petri dish with 400 μL of nuclei extraction buffer (CyStain ultraviolet Precise P Nuclei Extraction Buffer; Partec, Münster, Germany) using a sharp razor blade. The nuclei suspension was then filtered through 50-μm nylon filters and stained using 1.6 mL 4′, 6-diamidino-2-phenylindole (DAPI) staining buffer (Cystain ultraviolet Precise P Staining Buffer; Partec). Relative genome size was determined using a flow cytometer with fluorescence excitation provided by a mercury arc lamp (Partec PA-I; Partec). A minimum of 3000 nuclei was analyzed for each sample with a maximum cv of 4%, though it was typically below 3%. Pisum sativum ‘Ctirad’ (absolute 2C = 8.75 pg) was used as an internal standard for most taxa based on its common use as a reference standard (Bai et al., 2012; Greilhuber et al., 2007) and because its genome size was similar but distinct from most Cornus spp. As a result of the large relative genome size of C. eydeana, Magnolia virginiana ‘Jim Wilson’ (Moonglow™) was used as an internal standard (absolute 2C = 3.92 pg) for those analyses following Parris et al. (2010). Internal standards were cochopped with samples during nuclei extraction. Holoploid, 2C DNA contents were calculated as: 2C = DNA content of standard × (mean fluorescence value of sample ÷ mean fluorescence value of the standard). Analyses were completed on two subsamples for each replicate. The relationship between ploidy level and genome sizes was initially determined for species with documented chromosome numbers (Harrington et al., 1985; Li and Shang, 2002; Sandhu and Mann, 1988). Mean 1Cx monoploid genome size (i.e., DNA content of the non-replicated base set of chromosomes) was calculated as 2C genome size ÷ ploidy level.

Cytology.

Cytology was used to verify chromosome numbers and ploidy levels of eight taxa representing different taxonomic groups and to further calibrate genome sizes with ploidy levels (Table 1). Actively growing root tips, ≈10 mm in length, were collected from seedlings and rooted stem cuttings in the mornings before 1000 hr. Root tips were suspended in a pre-fixative solution of 2 mm 8-hydroxyquinoline + 0.24 mm cycloheximide in glass vials and stored in the dark at room temperature for 3 h. Root tips were then refrigerated at 6 °C for 3 h in the dark. After pre-fixative treatment, root tips were rinsed four times using refrigerated distilled H2O and then placed into a fixative solution of three parts 95% ethanol:one part propionic acid. Samples were then stored at room temperature overnight. The next morning, root tips were rinsed four times using 70% ethanol and stored in vials of 70% ethanol until needed.

Before squashing, root tip cell walls were hydrolyzed using three 95% ethanol:one 12 M hydrochloric acid for ≈3 min. After hydrolysis, root tips were then placed into a staining solution of modified carbol fuschin (Kao, 1975) for ≈5 min. The distal end of the stained root tip was excised under a dissecting microscope (Leica Stereozoom 6 Photo, Buffalo Grove, IL) and then placed on a glass microscope slide and gently squashed with a coverslip. Chromosomes were viewed using a light microscope (Nikon Eclipse 80i, Melville, NY).

Results and Discussion

Flow cytometry was an effective, efficient, and consistent method for determining relative genome sizes and ploidy levels of Cornus (Table 1). Values for the two subsamples of each replicate were consistent with sems for 2C values ranging from less than 0.01 to 0.2 pg with a mean of 0.05 pg. Few studies have reported genome sizes in Cornus, although Bai et al. (2012) reported the 2C value of one sample of C. canadensis as 4.4 pg [using propidium iodide (PI) stain] that is close to our findings that ranged from 4.2 to 4.3 pg (using DAPI stain). Zonneveld et al. (2005) found the 2C value of one sample of C. mas as 6.8 pg (using PI stain) similar to our values that ranged from 6.5 to 6.7 pg (using DAPI stain). Although different fluorochrome stains may give slightly different estimates of genome size (Doležel and Bartoš, 2005), both PI and DAPI have been found to be effective and consistent for determining and comparing ploidy levels and relative genome size among closely related taxa (Parris et al., 2010). Furthermore, the DAPI procedure is faster, less expensive, uses less toxic compounds, and typically produces results with a lower cv for mean nuclei fluorescence. Our estimates of 2C values for C. florida ranged from 3.05 to 3.32 pg, which was considerably higher than estimates of 2.19 to 2.77 pg reported by Wang et al. (2009), although both studies used DAPI stain.

Monoploid genome sizes (1Cx) varied among clades, subgenera, and species and ranged from 1.07 pg for C. canadensis to 5.16 pg for C. eydeana and were very consistent within species/grex with sems ranging from 0.01 to 0.12 pg and a mean sem of 0.03 pg (Table 2). Based on the taxa sampled, mean 1Cx values were smallest for the DW at 1.07 pg, the BB were intermediate at 1.89 pg, and the CC were considerably larger at 3.88 pg (Table 2).

There were also differences among subgenera within clades. For the BB dogwoods, mean 1Cx values for subgenus Syncarpea (2.00 pg) were significantly higher than for subgenus Cynoxylon (1.60 pg), whereas hybrids between these two subgenera were intermediate (1.83 pg). In some cases, there were also differences in 1Cx values among species within subgenera. For example, within subgenus Cornus, C. eydeana was significantly larger (5.08 pg) than C. mas (3.31 pg) and C. officinalis (3.28 pg). Also, within subgenus Cynoxylon, C. florida had a 1Cx mean of 1.58 pg, whereas C. nuttallii was significantly larger with a 1Cx mean of 1.71 pg. Monoploid genome sizes of evergreen species within Syncarpea (e.g., C. capitata, elliptica, and hongkongensis) varied some with C. capitata having the largest mean of 2.27 pg, and all of these species had significantly larger 1Cx values (2.07 to 2.27 pg) than that of C. kousa (1.92 pg). These results support the taxonomic groupings developed by Xiang et al. (2006) because the range of genome sizes for each of the subgenera and clades are distinct and discontinuous.

The only triploid identified in this study was the interspecific hybrid C. ‘KN30-8’ (NCSU 2012-008), [(C. kousa × C. nuttallii) × C. kousa], with 2n = 3x = 33 (Fig. 1C). It has been noted that hybrids often produce unreduced gametes at a particularly high frequency (Ramsey and Schemske, 1998), which may have contributed to the formation of this rare polyploidy in the BB clade. There has previously been some speculation that C. officinalis ‘Spring Glow’ may be a triploid based on its extremely low fruit set (personal observation), though its relative genome size is consistent with other diploids. It is also interesting to note that F1 hybrids of C. kousa × C. elliptica retain fertility (personal observation) though the parents vary in genome size by over 11%.

Relative genome sizes were also valuable for confirming hybridity of interspecific crosses in cases in which parents varied substantially in genome size (Fig. 2). Hybrids between species in the subgenera Syncarpea and Cynoxylon were readily apparent based on intermediate genome sizes including: C. capitata × C. florida, C. hongkongensis × C. florida, C. elliptica × C. florida, and C. ×rutgersensis (Table 2). In some cases, interspecific hybrids within subgenus Syncarpea could also be confirmed, including C. capitata × C. kousa and C. kousa × C. elliptica, though relative genome sizes were too similar between C. hongkongensis and C. kousa to verify hybrids between those species. Confirmation of these hybrids further suggests that there is considerable potential for the development of new hybrids among diverse species of BB dogwoods.

This study provides new and pertinent information on genome sizes and ploidy levels for species, cultivars, and hybrids of dogwoods. Additionally, it was found that genome size data can be an efficient and effective means of confirming hybridization among many BB dogwood species. These results provide further insights into the cytogenetics, systematics, reproductive biology, and crossability of dogwoods and contribute to the larger census of genome sizes of angiosperms.

KellerE.R.J.SchubertL.FuchsJ.MeisterA.1996Interspecific crosses of onion with distant Allium species and characterization of the presumed hybrids by means of flow cytometry, karyotype analysis and genomic in situ hybridizationTheor. Appl. Genet.92417424

Keller,E.R.J.Schubert,L.Fuchs,J.Meister,A.1996Interspecific crosses of onion with distant Allium species and characterization of the presumed hybrids by means of flow cytometry, karyotype analysis and genomic in situ hybridizationTheor. Appl. Genet.92417424)| false

XiangJ.Q.MoodyM.SoltisD.E.FanC.Z.SoltisP.S.2002Relationships within Cornales and circumscription of Cornaceae-matK and rbcL sequence data and effects of outgroups and long branchesMol. Phylogenet. Evol.243557

Xiang,J.Q.Moody,M.Soltis,D.E.Fan,C.Z.Soltis,P.S.2002Relationships within Cornales and circumscription of Cornaceae-matK and rbcL sequence data and effects of outgroups and long branchesMol. Phylogenet. Evol.243557)| false

XiangJ.Q.ThomasD.T.ZhangW.H.ManchesterS.R.MurrellZ.2006Species level phylogeny of the dogwood genus Cornus (Cornaceae) based on molecular and morphological evidence—Implication in taxonomy and Tertiary intercontinental migrationTaxon55930

Xiang,J.Q.Thomas,D.T.Zhang,W.H.Manchester,S.R.Murrell,Z.2006Species level phylogeny of the dogwood genus Cornus (Cornaceae) based on molecular and morphological evidence—Implication in taxonomy and Tertiary intercontinental migrationTaxon55930)| false

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Contributor Notes

This research was funded, in part, by the North Carolina Agricultural Research Service (NCARS), Raleigh, NC, the North Carolina Biotechnology Center, Research Triangle Park, NC, the Kenan Institute, Chapel Hill, NC, and the North Carolina Nursery and Landscape Association, Raleigh, NC.Technical assistance of Nathan Lynch, Joel Mowrey, Tom Eaker, Jeremy Smith, and Jason Lattier is gratefully acknowledged. We also thank Dr. Thomas Molnar at Rutgers University for providing samples of Cornus ‘KN30-8’ (Venus™).

KellerE.R.J.SchubertL.FuchsJ.MeisterA.1996Interspecific crosses of onion with distant Allium species and characterization of the presumed hybrids by means of flow cytometry, karyotype analysis and genomic in situ hybridizationTheor. Appl. Genet.92417424

Keller,E.R.J.Schubert,L.Fuchs,J.Meister,A.1996Interspecific crosses of onion with distant Allium species and characterization of the presumed hybrids by means of flow cytometry, karyotype analysis and genomic in situ hybridizationTheor. Appl. Genet.92417424)| false

XiangJ.Q.MoodyM.SoltisD.E.FanC.Z.SoltisP.S.2002Relationships within Cornales and circumscription of Cornaceae-matK and rbcL sequence data and effects of outgroups and long branchesMol. Phylogenet. Evol.243557

Xiang,J.Q.Moody,M.Soltis,D.E.Fan,C.Z.Soltis,P.S.2002Relationships within Cornales and circumscription of Cornaceae-matK and rbcL sequence data and effects of outgroups and long branchesMol. Phylogenet. Evol.243557)| false

XiangJ.Q.ThomasD.T.ZhangW.H.ManchesterS.R.MurrellZ.2006Species level phylogeny of the dogwood genus Cornus (Cornaceae) based on molecular and morphological evidence—Implication in taxonomy and Tertiary intercontinental migrationTaxon55930

Xiang,J.Q.Thomas,D.T.Zhang,W.H.Manchester,S.R.Murrell,Z.2006Species level phylogeny of the dogwood genus Cornus (Cornaceae) based on molecular and morphological evidence—Implication in taxonomy and Tertiary intercontinental migrationTaxon55930)| false